While adenoviral vectors continue to be employed in phase 1-III clinical trials for the treatment of cancers and cardiovascular diseases, we still have an incomplete understanding of the virus entry pathway. During the previous funding period, researchers in the Nemerow and Reddy laboratories at the Scripps Research Institute solved the crystal structure of adenovirus at near atomic resolution thereby revealing the locations, folds and interactions of several viral capsid proteins. Additional investigations in the Nemerow and Stewart laboratories (Vanderbilt) discovered that specific host factors either destabilize (alpha v integrins) the virus capsid or restrict (defensins) capsid disassembly. Although this information has shed further light on the mechanisms involved in virus entry into cells, numerous gaps remain in our knowledge of virus disassembly and membrane penetration events. In this proposal, a team of experienced investigators with expertise in molecular virology, structural biology, and biophysical techniques will investigate the precise mechanisms involved in capsid disassembly and membrane penetration by protein VI. X-ray diffraction and cryoEM structural analyses of several key capsid proteins including proteins VI, V and hexon will be undertaken. Advanced imaging of adenovirus by atomic force microscopy will be employed to measure the tensile strength and mechanical properties of the viral capsid. Native and denatured mass spectrometry will be used to determine the copy number of protein VI in the virion as well as its association states with other capsid proteins. Protein VI mutagenesis studies will analyze the role of protein VI in capsid assembly/disassembly and endosome penetration. The long-term goal of these studies is to obtain a more complete picture of host cell invasion by adenovirus. Such knowledge should bolster the emerging knowledge of how non-enveloped viruses breach host cell membranes as well as provide a foundation for the development of safer and more efficient viral vectors. PUBLIC HEALTH RELEVANCE: The use of viral vectors, including adenovirus, is stymied by a fundamental lack of knowledge of their structure and cell entry mechanisms. Inevitably, this situation negatively impacts implementation of effective vaccine, antiviral and gene delivery strategies. The specific goals of this proposal seek to remedy this deficiency by undertaking a comprehensive analysis of two key events in the adenovirus life cycle, capsid disassembly and membrane penetration.